Jay C. Erickson

Disruption of the Metallothionein-III Gene in Mice: Analysis of Brain Zinc, Behavior, and Neuron Vulnerability to Metals, Aging, and Seizures

Metallothionein-III (MT-III), a brain-specific member of the metallothionein family of metal-binding proteins, is abundant in glutamatergic neurons that release zinc from their synaptic terminals, such as hippocampal pyramidal neurons and dentate granule cells. MT-III may be an important regulator of zinc in the nervous system, and its absence has been implicated in the development of Alzheimer’s disease. However, the roles of MT-III in brain physiology and pathophysiology have not been elucidated. Mice lacking MT-III because of targeted gene inactivation were generated to evaluate the neurobiological significance of MT-III. MT-III-deficient mice had decreased concentrations of zinc in several brain regions, including hippocampus, but the pool of histochemically reactive zinc was not disturbed. Mutant mice exhibited normal spatial learning in the Morris water maze and were not sensitive to systemic zinc or cadmium exposure. No neuropathology or behavioral deficits were detected in 2-year-old MT-III-deficient mice, but the age-related increase in glial fibrillary acidic protein expression was more pronounced in mutant brain. MT-III-deficient mice were more susceptible to seizures induced by kainic acid and subsequently exhibited greater neuron injury in the CA3 field of hippocampus. Conversely, transgenic mice containing elevated levels of MT-III were more resistant to CA3 neuron injury induced by seizures. These observations suggest a potential role for MT-III in zinc regulation during neural stimulation.

Enhanced neurotrophic activity in Alzheimer's disease cortex is not associated with down-regulation of metallothionein-III (GIF).

Alzheimers disease (AD) is a chronic neurodegenerative disorder for which the pathogenic mechanisms are not well understood. Previous studies demonstrated that extracts prepared from AD brains could increase the survival of rat cortical neurons in vitro. Additional studies indicated that this enhanced neurotrophic activity of AD brain was due to a reduction of a growth inhibitory factor (GIF) that was subsequently shown to be a new member of the metallothionein (MT) gene family, and designated MT-III. The study presented here examined the association between neurotrophic activity and MT-III expression in frontal cortices from eight AD and five control brains, and further characterized the inhibitory activity of MT-III. On average, AD extracts stimulated the survival of approximately 2-fold more rat cortical neurons than control extracts, demonstrating that AD brain possesses elevated neurotrophic activity. When recombinant MTs were added to cultures grown in the presence of brain extract, MT-III but not MT-I had an inhibitory effect on neuron survival, confirming that MT-III is a specific inhibitory factor in this assay. However, in contrast to previous reports, neither MT-III mRNA nor MT-III protein levels were significantly decreased in the AD group. Therefore, the difference in neurotrophic activity between the AD and control brain samples examined in this study is probably not directly mediated by MT-III. These results suggest that MT-III down-regulation is not an important pathogenic event in some cases of AD.

Endocrine function of neuropeptide Y knockout mice

Among its many proposed functions, neuropeptide Y (NPY) is thought to modulate the hypothalamic-pituitary axis. Specifically, increased hypothalamic NPY signaling may be critical in mediating the neuroendocrine response to fasting. To determine the consequences of NPY deficiency on endocrine physiology, multiple hormones were quantitated in wildtype and NPY-knockout mice under fed and fasted conditions. Serum concentrations of leptin, corticosterone, thyroxine, and testosterone were normal in NPY-knockout males fed ad libitum. A 48-hour fast resulted in a 50% reduction in leptin, a 60% reduction in thyroxine, a 75% reduction in testosterone, and a 12-fold increase in corticosterone in both wildtype and NPY-knockout mice. Fasting also increased the estrous cycle length by 3 days in both wildtype and NPY-deficient female mice. We conclude that NPY is not essential for appropriate function of the gonadotropic, thyrotropic, or corticotropic axes under ad lib fed conditions or in response to acute fasting.

Response of neuropeptide Y-deficient mice to feeding effectors

Neuropeptide Y (NPY) is thought to be an important central regulator of feeding behavior and body weight. However, mice lacking NPY due to targeted genetic deletion do not display abnormalities in food intake or body weight with ad libitum access to food or in response to fasting. In this study, we investigate the response of NPY-deficient (NPY-/-) mice to anorexic and orexigenic treatments. The dose-dependent stimulation of food intake by central NPY administration was unaltered in NPY-/- mice. Peripheral administration of various doses of leptin for 2 days elicited a two-fold greater inhibition of food intake in NPY-/- mice than in wildtype (NPY+/+) mice. In addition, lateral ventricular administration of leptin (1 microg) suppressed refeeding in NPY-/- mice after a 24 h fast, but had little effect in NPY+/+ mice. However, the response to other feeding inhibitors such as corticotrophin releasing factor (CRF), dexfenfluramine, and a melanocortin 4 receptor (MC4R) agonist, MTII, was unaltered in NPY-/- mice. These results indicate that the appetite-suppressant action of exogenous leptin is uniquely amplified in NPY-/- mice, and suggest that NPY may tonically antagonize leptin action.

Role of neuropeptide Y in diet-, chemical- and genetic-induced obesity of mice

OBJECTIVE: The goal of this study was to ascertain whether neuropeptide Y (NPY) is required in mice for the development of obesity induced by a high-fat diet (HFD), chemical lesions of the hypothalamus caused by monosodium glutamate (MSG) or gold thioglucose (GTG), impaired brown adipose tissue (BAT) due to a diphtheria toxin transgene driven by the uncoupling protein 1 promoter (UCP-DTA) or the lethal yellow agouti mutation (Ay).BACKGROUND: The obesity syndrome of the leptin-deficient (ob/ob) mouse can be partially reversed by the genetic removal of NPY. In the murine models of obesity examined in this study, the animals become obese despite increased serum leptin levels, indicating that they are resistant to the weight-limiting actions of leptin. The role of NPY in these obesity models with elevated leptin levels is unknown.EXPERIMENTAL DESIGN: Mice lacking NPY due to genetic disruption of the gene and wildtype littermates were made obese by allowing them access to a highly palatable HFD, by treatment with MSG, or GTG, or by inheriting the dominant UCP-DTA or Ay alleles. Food consumption, body weight and dissectable fat pad weights were measured and compared to values obtained from non-obese littermates.RESULTS: In each model of obesity tested, NPY-deficient mice achieved the same food intake, body weight and fat content as wildtype littermates.CONCLUSION: NPY is not necessary for the progressive development of obesity exhibited by multiple murine models with leptin resistance.

Expression of human metallothionein-III in transgenic mice

Transgenic mice that express human metallothionein-III (hMT-III) were generated. Human MT-III mRNA expression was prominent in brain, resulting in a 9-fold elevation of MT-III mRNA in cortex, a 3-5-fold elevation in hippocampus, thalamus, brainstem, and olfactory bulb, and a 1.4-fold elevation in cerebellum. Human MT-III protein was detected biochemically and accounted for a 3.4-fold increase in total brain MT. The concentration of zinc (but not copper) was elevated in those brain regions that expressed the most hMT-III mRNA. The histochemically reactive pool of zinc, as measured by Timms stain or TS-Q histofluorescence, was not appreciably altered. No changes in brain weight, morphology or histology have been noted; the mice breed normally and appear to have normal behavior.

Effect of peripheral axotomy on dorsal root ganglion neuron phenotype and autotomy behaviour in neuropeptide Y-deficient mice

The lumbar 5 (L5) dorsal root ganglia (DRGs) were studied in neuropeptide tyrosine (NPY)-deficient (-/-) and wild type (+/+) mice after unilateral sciatic nerve transection using in situ hybridization and immunohistochemistry. NPY, galanin and two NPY receptors (Y-Rs) were analyzed as well as self-mutilation behaviour (autotomy) and nociceptive thresholds. No difference between wild type and NPY-deficient mice was seen in the tail-flick or hot plate test. However, -/- mice showed a much stronger autotomy behaviour than wild type mice. NPY was not found in L5 DRGs in -/- mice, not even after axotomy. Galanin was upregulated to the same extent after axotomy in NPY-deficient and wild type mice. Y1- and Y2-R mRNAs were found mainly in small DRG neuron profiles. Both receptor mRNAs were downregulated after axotomy, to about the same extent in NPY-deficient as in wild type mice. In control and contralateral ganglia the mRNA levels of both receptors were lower in NPY-deficient mice than in wild type mice. The contralateral Y2-R mRNA levels did not reach control values in the NPY-deficient mice, as they did in the wild type mice. In both strains the Y1-R protein was decorating the somatic plasmalemma. The present results suggest that lack of NPY may cause exaggerated autotomy, a self-mutilation behaviour possibly related to pain sensation, in agreement with the described analgesic effect of NPY. Although significant differences in levels of Y1- and especially Y2-R mRNAs were observed between wild type and NPY-deficient mice, they were only moderate. These findings suggest that expression, regulation, localization and possible function of Y1- and Y2-Rs are not dependent on presence of the endogenous ligand. Also, deletion of NPY does not seem to influence the expression of the partly coexisting peptide galanin.

The WWAMI Targeted Rural Underserved Track (TRUST) Program: An Innovative Response to Rural Physician Workforce Shortages.

PROBLEM Too few physicians practice in rural areas. To address the physician workforce needs of the Washington, Wyoming, Alaska, Montana, and Idaho (WWAMI) region, the University of Washington School of Medicine developed the Targeted Rural Underserved Track (TRUST) program in August 2008. TRUST is a four-year curriculum centered on a clinical longitudinal continuity experience with students repeatedly returning to a single site located in a rural community or small city. APPROACH The overarching theme of TRUST is one of linkages. Students are strategically linked to a rural community, known as their TRUST continuity community (TCC). The program begins with a targeted admission process and combines new and established programs and curricular elements to form a cohesive educational experience. This experience includes repeated preclinical visits, clerkships, and electives at a students TCC, and rural health courses, the Underserved Pathway, and the Rural Underserved Opportunities Program (which includes a community-oriented primary care scholarly project). OUTCOMES TRUST was piloted in Montana in 2008. With the matriculating class of 2015, every state in the WWAMI region will have TRUST students. From 2009 (the year targeted admissions began) to 2015, 123 students have been accepted into TRUST. Thirty-three students have graduated. Thirty (90.9%) of these graduates have entered residencies in needed regional specialties. NEXT STEPS Next steps include implementing a robust evaluation program, obtaining secure institutional programmatic funding, and further developing linkages with regional rural residency programs. TRUST may be a step forward in addressing regional needs and a reproducible model for other medical schools.

The growth inhibitory activity of metallothionein-3 correlates with its novel β domain sequence rather than metal binding properties

A neuronal survival assay consisting of cultured neonatal rat cortical neurons supplemented with Alzheimer brain extracts was used to identify a protein designated growth inhibitory factor (GIF) as a candidate growth inhibitory component in normal brain tissue [1]. Subsequent characterization of GIF revealed a 70% sequence homology with mammalian metallothionein (MT) including the 20 metal-binding cysteine residues in conserved Cys-X-Cys and Cys-X-XCys motifs. The GIF gene also has a similar intron/exon structure to that of mammalian MT-1 and MT-2 leading to its classification as MT-3 [2]. Relative to mammalian MTs, MT-3 contains two inserts, a single threonine residue in the N-terminal β-domain and a glutamine rich hexapeptide in the C-terminal α-domain.